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Posted by jkrise on 24 Apr 2012 at 21:32 GMT
This was a very interesting and thought-provoking article. I very much admire the approach the authors taking to understand the potential therapeutic mechanisms of these types of drugs. Please see some specific questions and comments that I have regarding this work below.
1) In Figure 2A you examined the effect of reduced temperature on the accumulation of seratraline. You have attributed the decrease at lower temperatures to the decrease in membrane fluidity, which could decrease the partitioning of the drug in the membrane. I agree with this possibility but isn’t it also a possibility that the activity of the V-ATPase could be compromised at lower temperature and that this could account for some of the differences?
2) The finding that energy poisons significantly increased the total cellular accumulation of a lysosomotropic drug is interesting. Lysosomal uptake and cellular efflux are both energy dependent processes. This result highlights the significant role of efflux transporters in yeast.
3) In figure 3A, most of the drug is associated with the pellet but do you think that the organelles are intact when pelleted (i.e., would they contain soluble drug as well as membrane associated)? Perhaps reconstituting the pellet in pure water might osmotically rupture them without interfering with membrane composition, thus allowing you to tease these apart.
4) Considering the potentially important role of the efflux transporters in regulating the accumulation of Sert in yeast (Figure 2C), is it possible that some of the mutants could be influencing the activity and/or cellular trafficking of these transporters? Of specific interest is the swa2 mutant since doesn’t appear to be sensitive to BAF treatment suggesting that the Sert is not extensively accumulated via ion trapping mechanism in the vacuole when associated with cells. However, the total cellular accumulation of the Sert in the swa2 mutant is greater than in wild type yeast. This would appear to be consistent with the swa2 mutant having decreased activity of efflux transporters as well as decreased accumulation in vacuoles. Is this consistent with your thinking?
5) In the discussion you imply that the ionized pool of sert is actively depleted by transporter mediated efflux and by sequestration in the cellular membranes as the neutral species. I seem to have some difficulty interpreting this statement. It would seem logical to me that proton transfer (i.e., re-establishment of the percentage of drug that is ionized at any given time) would be much faster than any drug transporter or membrane binding event so I am not sure how these activities could specifically deplete the ionized pool of drug without depleting the unionized pool as well. On a related note, you appear to imply that Sert is associated with membranes as the neutral species. When associated with the membrane I would think that it would exist both ionized and unionized depending on the apparent pKa of membrane-bound Sert, which may be somewhat different than Sert in an aqueous environment. The only way that I see it being substantially neutral when bound to membranes is if the pKa of Sert were to drop to values below the pH of the acidic vacuolar lumen (i.e., pKa 3 or lower). Could you please elaborate?
6) How do you think that autophagy can physically decreases CAD accumulation in membranes?
Thanks for the refreshingly incisive comments. Please see my answers below. (Again much appreciation for actually reading the paper!)
A1: That is certainly possible, and would be consistent with your published results in human cells. I admit that the membrane fluidity claim requires much more rigorous testing.
A2: I agree. Cellular efflux is dominant to lysosomal uptake. I think it explains why so many yeast-based chemical screens employ drug-sensitized efflux deletion mutants, e.g., pdr5, snq2.
A3: We see pretty much the same distribution when we lysed the cells mechanically or non-mechanically, i.e., with detergent (Fig 3A). But I agree with your experimental suggestion and we'll get going on trying it in the lab, so stay tuned!
A4: It is possible, though it doesn't explain the modest but specific sub-lethal trophic effect of sertraline on the chc1 and swa2 mutants, which we first observed in our 2010 Genetics paper (http://www.genetics.org/c...). Especially in light of the fact that the original pressure that selected for these mutations was drug overdose. Normally, knocking out drug efflux causes drug hypersensitivity, not the resistance which we observed. We would interpret the result this way: the hyperaccumulation of sertraline by swa2 and chc1 mutants could be explained by the increased lysosomal membrane surface area to volume ratio based on the vacuolar fragmentation phenotype we observe by TEM (Fig. 5C,D). Now, you rightly point out that we are not observing the precise in vivo sertraline equilibration in our lysis procedure. We are attempting autoradiography experiments to sort all this out directly. Those (challenging) experiments are now underway in my lab.
A5: I admit that my interpretative powers are at a nadir on the question of precise bilayer localization, but I agree with your assessment. I was simply trying to channel the spirit of the Journal of Biophysics paper on tetracaine-membrane partitioning (ref #20). I'm actually in discussions like a potential collaborator to address the biophysical aspects of drug-membrane interactions. The more carefully I've read up in this area, the more I've come to appreciate the subtleties at play here. However, I've mostly come up with many interesting but ultimately removed biophysics studies. If you can point me to good experimental literature on pKa of native membrane-associated cationic amphipaths, I would be appreciative. My lab is collaborating with Sepp Kohlwein's group to chase down our preliminary findings on the acute effects of sertraline on lipid droplet homeostasis, which we think is a place where sertraline may be specifically accumulating.
A6: If that was the impression you got, it was not what I meant! I assume that you're referring to the Discussion? My point was that the membrane curvature stress is "relieved" by autophagy in a physiological sense. I was not arguing that autophagy leads to efflux.
I also enjoyed reading this interesting article. From the basic Chemistry point of view, there are 2 factors that are important: the charge and amphipathicity of the molecule. A previous NMR study demonstrated the positive charge generated on the model membrane due to the interaction of an antidepressant, desipramine, is the key factor in its mechanism.
1) Interestingly, this study showed that the uptake of radiolabeled Zoloft depends on extracellular pH and organellar pH in a manner consistent with lysosomotropism. So, acidic external pH reduces the uptake potential because nearly all the Zoloft is ionized and therefore membrane-impermeable; and disrupting the proton gradient across organellar membranes (where the V-ATPase is expressed) also reduces uptake potential.
2) This study also showed biochemically that radiolabeled Zoloft pellets with large organellar membranes from cellular lysates, and can be extracted from these membranes by detergents.